WO2021117915A1 - Scaffold using adipose tissue-derived extracellular matrix, and method for producing same - Google Patents

Abstract

The present invention relates to an allogeneic and heterogeneous adipose tissue-derived extracellular matrix scaffold, and a method for producing same. An adipose tissue-derived extracellular matrix scaffold according to the present invention has a composition similar to the human body, a large surface area, and an interconnected porous structure, and thus has high cell affinity and allows cells to survive for long periods of time.

Description

Support using adipose tissue-derived extracellular matrix and method for manufacturing the same

The present invention relates to an extracellular matrix scaffold derived from allogeneic and heterogeneous adipose tissue and a method for preparing the same. More specifically, the present invention relates to an extracellular matrix derived from adipose tissue derived from allogeneic and heterogeneous adipose tissue that has components similar to those of the human body, has a large surface area, and has a porous structure interconnected, high in cell affinity, and in which cells can survive for a long time and It relates to a manufacturing method.

Regenerative medicine aims to replace or regenerate human cells, tissues, and organs. Traumatic trauma that causes tissue damage and loss of function, and the emergence of new diseases in accordance with the advancement of society, provide an inevitable motive for the rapid development of the field of regenerative medicine.

Medical materials used in the field of regenerative medicine should be carefully selected according to the type of tissue and organ to be applied, the type of disease or trauma, and the patient's medical history. In general, the most frequently selected materials for research include heterogeneous extracted collagen and gelatin, microorganism-derived hyaluronic acid, chitosan, vegetable cellulose-based polymers, vegetable alginate, and the like. In addition, allogeneic substances that can be obtained from human cadavers are attracting attention as effective biomaterials that can be safely used in the field of regenerative medicine.

Among biomaterials, adipose tissue, as well as safety and effectiveness as a biomaterial, is gaining economic/industrial interest both at home and abroad. Adipose tissue is a loose connective tissue composed of adipocytes, preadipocytes, fibroblasts, vascular endothelial cells, and various immune cells. Adipose tissue contains extracellular matrix such as collagen, elastin, laminin, fibronectin, glucosaminoglucan and the like. The extracellular matrix not only helps support and proliferation of cells in the tissue in vivo, but also helps in the recovery of damaged areas in the living body by maintaining the tissue by binding to the cells.

Allogeneic and heterogeneous adipose tissue-derived extracellular matrix has been studied as a support for replacement and reinforcement of damaged human tissue and for cell culture. Recently, in preclinical experiments, it has been reported that adipose tissue-derived extracellular matrix scaffolds are effective in repairing defective tissues. In addition, it has been reported that the adipose tissue-derived extracellular matrix support affects the growth of cells, which is due to the porous structure and components.

However, these homogeneous and heterogeneous adipose tissue-derived extracellular matrix scaffolds are generally prepared using a combination of surfactants and enzymes. It even inhibits the growth of the target cell. In addition, it has a disadvantage that a long-term manufacturing process is required.

[Prior art literature]

[Patent Literature]

1. Republic of Korea Patent 10-0771058

2. Republic of Korea Patent Registration 10-1628821

[Non-patent literature]

1. Combining decellularized human adipose tissue extracellular matrix and adipose-derived stem cells for adipose tissue engineering, Acta Biomaterialia 2013, 8921-31

2. Biocompatibility of injectable hydrogel from decellularized human adipose tissue in vitro and in vivo, Journal of Biomedical Materials Research Part B, 2018, 1684-1694

3. The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells, Biomaterials, 2010, 4715-24

4. Simulation of tissue differentiation in a scaffold as a function of porosity, Young's modulus and dissolution rate: Application of mechanobiological models in tissue engineering, Biomaterials, 207, 5544-5554

Accordingly, in the present invention, it is to provide an adipose tissue-derived extracellular matrix scaffold that has components similar to those of the human body, has a large surface area, has a interconnected porous structure, and thereby has high cell affinity and enables cells to survive for a long time, and a method for manufacturing the same The purpose.

More specifically, an object of the present invention is to provide an adipose tissue-derived extracellular matrix support and a manufacturing method that can achieve high cell affinity with low toxicity, induce self-organization, shorten the manufacturing period, and realize low manufacturing cost.

The present invention provides a de-fat step to remove a lipid component from adipose tissue;

a decellularization step of removing cells from the adipose tissue from which the lipid component has been removed; and

It comprises a freeze-drying step of freeze-drying the adipose tissue from which the cells have been removed,

The decellularization step provides a method for preparing adipose tissue-derived extracellular matrix scaffolds performed using a basic solution.

In addition, the present invention provides an adipose tissue-derived extracellular matrix support prepared by the above-described production method.

The present invention provides a novel manufacturing method for preparing an adipose tissue-derived extracellular matrix scaffold. Conventionally, it took about 7 to 10 days to prepare the adipose tissue-derived extracellular matrix scaffold, but when the manufacturing method according to the present invention is used, the period can be shortened to 3 days or less.

In addition, it is possible to induce the porous structure to be well maintained in the extracellular matrix by using a basic solution during decellularization, and to contain the active ingredient of adipose tissue. And, it is possible to provide an extracellular matrix support through which the cell affinity is improved and the cells can survive for a long time.

1 is a photograph of various types of extracellular matrix support according to an example of the present invention.

Figure 2 is a photograph of performing Oil Red O staining in order to confirm the residual amount of fat in the extracellular matrix support according to an example of the present invention.

3 is a graph showing a photograph of DAPI staining and a quantification of the DNA content in order to confirm the remaining amount of cells in the extracellular matrix support according to an example of the present invention.

4 is a photograph taken with a scanning electron microscope to analyze the structure of the extracellular matrix scaffold according to an example of the present invention.

5 is a photograph confirmed by the Live/dead cell viability assay kit to analyze the growth of cells in the extracellular matrix support according to an example of the present invention, and a graph quantifying it.

The present invention provides a de-fat step to remove a lipid component from adipose tissue;

a decellularization step of removing cells from the adipose tissue from which the lipid component has been removed; and

It relates to a method for producing an adipose tissue-derived extracellular matrix scaffold comprising a freeze-drying step of freeze-drying the adipose tissue from which the cells have been removed.

In an embodiment of the present invention, an adipose tissue-derived extracellular matrix scaffold is prepared through the steps according to the invention, and the porosity is uniform and the structure does not collapse compared to the comparative example, an extracellular matrix support prepared using a conventional surfactant and enzyme. It was confirmed that the support was prepared, and it was confirmed that the survival and growth of cells in the support were excellent.

Hereinafter, the method for producing the adipose tissue-derived extracellular matrix scaffold of the present invention will be described in more detail.

The method for producing an adipose tissue-derived extracellular matrix scaffold (hereinafter, referred to as an extracellular matrix support) of the present invention includes a de-lipidation step; decellularization step; and freeze-drying.

In one embodiment, the extracellular matrix (ECM) refers to a complex aggregate of biopolymers filling the tissue or extracellular space. The components of the extracellular matrix may vary depending on the type of cell or the degree of differentiation of cells, and fibrous proteins such as collagen and elastin, complex proteins such as proteoglycans and glycosaminoglycans, and fibronectin, laminin, etc. of cell-adherent glycoproteins.

In one embodiment, the adipose tissue may be allogeneic or heterogeneous adipose tissue. The same species refers to humans, and the heterogeneous species refers to animals other than humans, that is, mammals such as pigs, cattle, and horses, as well as fish.

That is, in the present invention, the extracellular matrix can be prepared according to the preparation method of the present invention using adipose tissue derived from allogeneic or heterogeneous.

The present invention may perform a washing step prior to performing the defatting step. In the washing step, the adipose tissue may be washed with sterile distilled water. Through the above step, impurities in the adipose tissue can be removed.

In the present invention, the delipidation step is a step of removing the lipid component from the adipose tissue.

In one embodiment, delipidation refers to the removal of a lipid component from a tissue.

In one embodiment, the removal of the lipid component may be performed by physical treatment or chemical treatment, and the physical treatment and chemical treatment may be performed together. When performed together, the order of execution is not limited.

In one embodiment, the type of physical treatment is not particularly limited and may be performed using pulverization. The pulverization may be performed using a pulverizing means known in the art, for example, a mixer, a homogenizer, a frozen pulverizer, an ultrasonic pulverizer, a hand blender, a plunger mill, and the like.

When performing the pulverization, the pulverized product, that is, the pulverized adipose tissue may have a particle diameter of 0.01 to 1 mm.

In one embodiment, the type of chemical treatment is not particularly limited and may be performed using a delipidation solution. The delipidation solution may include a polar solvent, a non-polar solvent, or a mixed solvent thereof. Water, alcohol, or a mixed solution thereof may be used as the polar solvent, and methanol, ethanol or isopropyl alcohol may be used as the alcohol. As the non-polar solvent, hexane, heptane, octane, or a mixed solution thereof may be used. Specifically, in the present invention, a mixed solution of isopropyl alcohol and hexane may be used as the delipidation solution. In this case, the mixing ratio of isopropyl alcohol and hexane may be 40:60 to 60:40.

The treatment time of the delipidation solution may be 4 to 30 hours, or 10 to 20 hours.

In one embodiment, the delipidation step may be performed by sequentially applying a physical treatment and a chemical treatment. The lipid component may be primarily removed from the adipose tissue by physical treatment, and the lipid component not removed by the physical treatment may be removed by chemical treatment.

In the present invention, the decellularization step is a step of removing cells from the adipose tissue from which the lipid component has been removed by the delipidation step.

In one embodiment, decellularization refers to the removal of other cellular components other than the extracellular matrix from a tissue, for example, a nucleus, a cell membrane, a nucleic acid, and the like.

In one embodiment, decellularization may be performed using a basic solution, and one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, calcium hydroxide and ammonia may be used as the basic solution. have. In the present invention, sodium hydroxide (NaOH) may be used as the basic solution. Conventionally, decellularization was performed using a surfactant and an enzyme. However, in this case, the final prepared extracellular matrix supporter does not maintain porosity in its structure, and has a problem in that the growth of cells is inhibited. In the present invention, the above problems can be solved by using a basic solution during decellularization, and there is an advantage that there is no cytotoxicity.

In one embodiment, the concentration of the basic solution may be 0.01 to 1 N, 0.06 to 0.45 N, 0.06 to 0.2 N, or 0.08 to 1.02 N. In the above concentration range, it is easy to remove cells, and an extracellular lipid scaffold having a structure in which pores are connected and does not collapse can be prepared.

Also, in one embodiment, the decellularization step may be performed for 60 to 48 minutes, 70 to 200 minutes, or 90 to 150 minutes. In the above time range, it is easy to remove cells, and an extracellular lipid scaffold having a structure in which pores are connected and does not collapse can be prepared.

In the present invention, after performing the decellularization step, a centrifugation step may be additionally performed before performing the freeze-drying step. Through the centrifugation step, impurities in the delipidation step and the decellularization step can be removed, and a high-purity extracellular matrix material (precipitate) can be obtained.

In one embodiment, centrifugation may be performed at 4,000 to 10,000 rpm, or 8,000 rpm for 5 to 30 minutes, 5 to 20 minutes, or 10 minutes.

In addition, before and/or after centrifugation, a washing step may be additionally performed, and sterile distilled water may be used for washing.

In the present invention, the freeze-drying step is a step of freeze-drying the obtained product after the aforementioned step, that is, the decellularization step or the centrifugation step. The freeze-drying is a method of rapidly cooling the tissue in a frozen state and then absorbing moisture in a vacuum. By performing the freeze-drying, moisture in the extracellular matrix material can be controlled, and the extracellular matrix support having a porous structure interconnected can be manufactured with

In one embodiment, freeze-drying may be performed at -50 to -80°C for 24-96 hours.

In one embodiment, the moisture content of the lyophilized extracellular matrix scaffold may be 10% or less, or 1 to 8%.

In the present invention, after performing the freeze-drying step, a sterilization step of sterilizing the extracellular matrix support may be additionally performed. Through the sterilization step, the immunity in the extracellular matrix can be removed, and bacteria can be effectively destroyed.

In one embodiment, the sterilization step may be performed by irradiating radiation, and the irradiation range of radiation may be 10 to 30 kGy.

In addition, the present invention is a washing step of washing the adipose tissue;

a de-fatting step of removing lipid components from the washed adipose tissue;

a decellularization step of removing cells from the adipose tissue from which the lipid component has been removed;

a centrifugation step of centrifuging the decellularized adipose tissue;

a freeze-drying step of freeze-drying the precipitate after centrifugation; and

It relates to a method for producing an adipose tissue-derived extracellular matrix support comprising a sterilization step of sterilization.

The step may be performed as described above.

In addition, the present invention relates to an adipose tissue-derived extracellular matrix scaffold prepared by the above-described method for producing an adipose tissue-derived extracellular matrix scaffold.

In one embodiment, the water content of the extracellular matrix scaffold may be 10% or less.

Further, in one embodiment, the porosity of the extracellular matrix scaffold may be 10 μm to 800 μm, 100 to 500 μm, and the porosity may be 30 to 80%, or 40 to 60%.

The extracellular matrix support may have a component similar to that of a human body, have a large surface area, and have an interconnected porous structure. Therefore, the extracellular matrix scaffold of the present invention has high cell affinity, and the cells can survive for a long time. Therefore, the extracellular matrix scaffold can be used as a scaffold for replacement and reinforcement of damaged human tissue and cell culture.

The present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and it will be understood by those skilled in the art that various changes, modifications or applications are possible within the scope without departing from the technical matters derived from the matters described in the appended claims. will be.

Example

Example 1. Preparation of human adipose tissue-derived extracellular matrix scaffold

Human adipose tissue was pulverized with a grinder to remove fat. In order to remove the fat that did not fall off, it was subjected to a de-fat process for 16 hours using 40% to 60% isopropyl alcohol and 40% to 60% hexane. The cells were removed by treatment with 0.01 to 1N sodium hydroxide (NaOH) in the fat-removed tissue.

The supernatant was removed by centrifugation at 8,000 rpm for 10 minutes to wash the extracellular matrix from which the fat and cells were removed, and the washing process was repeated 5 to 10 times. After freeze-drying the support so that the moisture content of the human adipose tissue-derived extracellular matrix is 10% or less, preferably 1% to 8%, radiation sterilization was performed to prepare an extracellular matrix support.

Table 1 shows the results of observing the change of the prepared extracellular matrix support according to the treatment time after the adipose tissue was supported in sodium hydroxide at various concentrations during the decellularization process.

Through the results in Table 1, the optimal concentration (0.1N) and time (2 hrs) of sodium hydroxide treatment can be confirmed. It is easy to remove cells at the above concentration and time, and it is possible to prepare an extracellular lipid scaffold having a structure in which pores are connected to each other and do not collapse.

In addition, Figure 1 is a photograph of the support prepared in Example 1.

As shown in FIG. 1, it can be seen that the extracellular matrix scaffold prepared by the method of the present invention has a large surface area and a porous structure interconnected.

Experimental Example 1. Confirmation of residual fat in human adipose tissue-derived extracellular matrix scaffold

(1) method

The human adipose tissue-derived extracellular matrix scaffold prepared by the method of Example 1 was used as an experimental group and adipose tissue as a control group.

Oil Red O staining was performed to evaluate the residual fat of the extracellular matrix scaffold.

(2) Results

The results of performing Oil Red O staining are shown in FIG. 2 .

As shown in FIG. 2 , it can be confirmed that fat has been removed from the human adipose tissue-derived extracellular matrix scaffold prepared by the method of Example 1.

Experimental Example 2. Confirmation of residual cells in human adipose tissue-derived extracellular matrix scaffolds

(1) method

The human adipose tissue-derived extracellular matrix scaffold prepared by the method of Example 1 was used as an experimental group and adipose tissue as a control group.

DAPI staining was performed to qualitatively evaluate residual cells. In addition, DNA content was measured to quantitatively evaluate residual cells.

(2) Results

The residual cell measurement results are shown in FIG. 3 .

In FIG. 3, 3A shows a photograph of DAPI staining, and 3B shows a graph quantifying the DNA content.

As shown in FIG. 3, it can be confirmed that cells are removed from the human adipose tissue-derived extracellular matrix scaffold prepared by the method of Example 1, and the DNA content of the extracellular matrix support is 50 ng/mg or less. that can be checked

Comparative Example 1.

An extracellular matrix scaffold derived from human adipose tissue was prepared by a conventional method (surfactant and enzyme).

First, human adipose tissue was washed for 2 days. To further wash the fat, it was treated with 0.5 N NaCl for 4 hours and 1N NaCl for 4 hours. The washed adipose tissue was treated with 0.25% trypsin (enzyme) and EDTA for 2 hours.

The enzyme-treated adipose tissue was treated with 100% isopropyl alcohol for 16 hours to defat. To further remove cells, 1% trypsin was treated for 3 days.

After removal of fat and cells, the extracellular matrix was washed for 2 days. After freeze-drying the support so that the moisture content of the human adipose tissue-derived extracellular matrix is 10% or less, preferably 1% to 8%, radiation sterilization was performed.

Experimental Example 3. Confirmation of functions of extracellular matrix support derived from human adipose tissue

3-1. Scanning electron microscope of human adipose tissue-derived extracellular matrix scaffold

(1) method

The human adipose tissue-derived extracellular matrix support prepared by the method of Example 1 was used as an experimental group, and the human adipose tissue-derived extracellular matrix support prepared by the method of Comparative Example 1 was used as a control group.

By photographing through a scanning electron microscope, the porous structure of the extracellular matrix scaffolds of Example 1 and Comparative Example 1 was analyzed.

(2) Results

The porous structure analysis result is shown in FIG. 4 . 4 shows a photograph taken with a scanning electron microscope.

As shown in FIG. 4, Comparative Example 1, that is, the extracellular matrix support prepared by the control has non-uniform porosity and the structure collapses, but the human adipose tissue-derived extracellular matrix support prepared by the method of Example 1 can be qualitatively confirmed that the porosity is uniform, has a connected pore structure, and the structure does not collapse.

2-2. Confirmation of cell growth in human adipose tissue-derived extracellular matrix scaffold

(1) method

In the cell growth experiment in the extracellular matrix scaffold, the human adipose tissue-derived extracellular matrix support prepared by the method of Example 1 was used as an experimental group, and the human adipose tissue-derived extracellular matrix support prepared by the method of Comparative Example 1 was used as a control group. carried out.

1×10 ⁵ cells/100 μl of fibroblasts were dispensed on the support and immersed in a culture medium to proceed with culturing.

To evaluate cell growth, 1, 7, and 14 days after culture were stained with Live/dead cell viability assay kit (Life Technology, USA). A medium in which 0.5 μl/ml Calcein-AM and 2 ul/ml Ethidium homodimer-1 were dissolved was immersed in the construct and reacted for 30 minutes. After the reaction, it was confirmed through a confocal microscope (LSM 700, Carl Zeiss, Germany). Cell survival inside the construct was confirmed by focusing at intervals of 10 μm to a depth of about 200 μm.

(2) Results

The cell growth confirmation result is shown in FIG. 5 .

5 is a photograph and quantitative graphs confirmed by the Live/dead cell viability assay kit to analyze the growth of cells.

As shown in FIG. 5 , it can be seen that the number of living cells increased over time in the human adipose tissue-derived extracellular matrix scaffold prepared by the method of Example 1 compared to Comparative Example 1, that is, the control group. In addition, through the graph, it can be confirmed that the number of cells increased by 5 times or more compared to the control on the 14th day of culture.

The extracellular matrix scaffold according to the present invention may have a component similar to that of a human body, have a large surface area, and have a porous structure interconnected. Therefore, the extracellular matrix scaffold of the present invention has high cell affinity, and the cells can survive for a long time. Therefore, the extracellular matrix scaffold can be used as a scaffold for replacement and reinforcement of damaged human tissue and cell culture.

Claims (11)

1. a delipidation step of removing lipid components from adipose tissue;

   a decellularization step of removing cells from the adipose tissue from which the lipid component has been removed; and

   It comprises a freeze-drying step of freeze-drying the adipose tissue from which the cells have been removed,

   The decellularization step is performed using a basic solution

   A method for preparing an adipose tissue-derived extracellular matrix scaffold.
2. The method of claim 1,

   Adipose tissue is a method for producing an adipose tissue-derived extracellular matrix scaffold, which is an allogeneic or heterogeneous adipose tissue.
3. The method of claim 1,

   A method for producing an adipose tissue-derived extracellular matrix scaffold, wherein the removal of the lipid component is performed by physical treatment and/or chemical treatment.
4. 4. The method of claim 3,

   A method for producing an adipose tissue-derived extracellular matrix scaffold, wherein the physical treatment is pulverization.
5. 4. The method of claim 3,

   Chemical treatment is carried out using a delipidation solution,

   The delipidation solution is a method for producing adipose tissue-derived extracellular matrix support comprising a polar solvent, a non-polar solvent, or a mixed solvent thereof.
6. The method of claim 1,

   The basic solution is sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium carbonate, magnesium hydroxide, a method for producing adipose tissue-derived extracellular matrix scaffold comprising at least one selected from the group consisting of calcium hydroxide and ammonia.
7. The method of claim 1,

   The concentration of the basic solution is 0.01 to 0.1 N,

   A method for producing an adipose tissue-derived extracellular matrix scaffold, wherein the treatment time is 60 to 480 minutes.
8. The method of claim 1,

   After performing the decellularization step, a method for producing an adipose tissue-derived extracellular matrix scaffold further comprising the step of centrifugation.
9. The method of claim 1,

   The freeze-drying step is a method for producing adipose tissue-derived extracellular matrix scaffolds that are performed at -50 to -80 ℃ for 24 to 96 hours.
10. The method of claim 1,

    After performing the freeze-drying step, a method for producing an adipose tissue-derived extracellular matrix scaffold to further perform a sterilization step.
11. It is prepared by the manufacturing method according to claim 1,

    Adipose tissue-derived extracellular matrix support with a water content of 10% or less.

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